PROJECTSUMMARY/ABSTRACT Tissuemanufacturingforskinreplacementtherapyisextremelyinefficient,inpartduetoanincomplete understandingofgeneexpressionmechanismsregulatingstemcellcommitment.Onesuchpoorlyunderstood mechanismischromatinlooping,whichhasbeenthoughttoserveasatemplateforgeneexpressionchanges duringcellstatetransitions.Thisknowledgegapisamajorroadblockinproducingsufficientgraftable keratinocytesfromgeneticallycorrectedpatientstemcellsfortreatmentofdebilitatingskindiseases.Thelong- termgoalofthisproposalistounderstandthemolecularmechanismsunderlyingstemcelldifferentiationto improveskinreplacementtherapy.Preliminarydatafromtheapplicant?sgroupsuggestthatretinoicacid(RA) andbonemorphogenicprotein(BMP4)morphogenscaninducestemcell-derivedgraftablekeratinocytes throughinductionofthemasterepithelialregulatorandtranscriptionfactorp63.Interestingly,p63cannot activatedownstreamgeneexpressionprogramsintheabsenceofthesemorphogens.Further,high dimensionalchromatinanalysessuggestthatmorphogenscausemajorchangesinchromatinlooping. Therefore,thecentralhypothesisisthatRAandBMP4stimulateloopingbetweenp63bindingsitesanddistal loci,aswellasalterthep63interactome,tofacilitatep63-dependentgeneexpression.Thisproposalwilltest thehypothesisbypursuingtwospecificaims.Aim1willdeterminewhichregulatoryregionswithinchromatin loopsarerequiredforp63-dependentgeneexpression.CRISPR/Cas9tools,whichhavebeenwellestablished intheapplicant?slaboratory,willbeusedtosequentiallyblockp63bindingsitesandthelocitowhichtheyare looped.Theeffectsofeachdeletionwillbemeasuredbydownstreamgeneexpression,loopformation,and cellulardifferentiationmarkers.TheproposedexperimentswillfocusonloopsatTFAP2CandHES1loci, whichareknowntobecrucialduringstemcellcommitment.Aim2willelucidatep63interactingproteinsthat arerequiredfordownstreamgeneexpression.Candidatemembersofthep63interactomewillbeidentified usinganovelproximitylabelingsystemknownasBASU.Thissystemhasbeenvalidatedintheapplicant?s laboratoryusingembryonicstemcells.Theimportanceofeachp63interactingproteinwillthenbeevaluated usingaCRISPR/Cas9lossoffunctionscreenfollowedbymeasurementsofp63-dependentgeneexpression andcellulardifferentiation.Therationalefortheproposedresearchisthatitwillprovideusefulchromatin dynamicinformationwhichcanbeusedtoimprovethecurrentstemcelldifferentiationprotocolfortissue replacementtherapy.Thisproposalisinnovative,becauseitusesnovelgeneticandproteomicstechniquesto elucidatemechanismsofchromatinloopsthatwerepreviouslyunrecognized.Thefindingswillbesignificant, becausetheywillbroadentheunderstandingofp63duringdevelopmentaswellascontributetomuchneeded improvementsforskinreplacementtherapy.Takentogether,thisknowledgewillprovidecrucialinformationon howchangesinchromatinarchitecturecangeneratediversityinmorphologicalpatterning.